Bs 0169r

The cells were washed twice with cold PBS buffer and lysed on ice with RIPA (P0013B, Beyotime, China) lysis buffer for 30 min. The lysate was centrifuged at 4°C for 15 min at 14,000 g. The supernatant was collected, and protein was extracted by the Beyotime BCA kit (P0010, Beyotime, China) to determine the protein concentration. The protein was then denatured in loading buffer and denatured by boiling in a 95°C water bath for 5 min. The protein samples (20 μg of protein per gel lane) were loaded onto SDS–PAGE gels and then transferred to PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) for 2 h at room temperature and incubated overnight in the primary antibody at 4°C. The antibody information is as follows: LC3 (#3868 s, Cell Signaling Technology, USA), Lamp1 (ab24170, Abcam, UK), NLRP3 (bs-10021R, Bioss, China), Caspase-1 (#3866, Cell Signaling Technology, USA), Caspase-1 (bs-0169R, Bioss, China), IL-1β (bs-0812R, Bioss, China), TFEB (ab270604, Abcam, UK), and β-actin (AA128-1, Beyotime, China). The PVDF membrane was washed and incubated with horseradish peroxidase and secondary antibody for 1 h. An enhanced chemiluminescence kit (BL520B, Biosharp, China) and gel imaging system (Bio-Rad, USA) were utilized to collect protein development images. ImageJ software was used for the quantitative analysis of signals.